Nucleoside triphosphate compounds can be polymerized to provide ribonucleic acid (RNA) and deoxyribonucleic acid (DNA). Apart from the traditional role in normal cells, these nucleic acids form the genetic materials of a variety of pathogenic viruses such as HIV, herpes, measles, mumps and many others. Many nucleoside triphosphates are not particularly robust, and they also tend to be difficult to synthesize. Many methods involve difficult combinations of charged ionic reagents with more lypophilic substrates, such as combinations of pyrophosphates and protected nucleosides. According to Burgess, et al., Chem. Rev., 2000, 100, 2047-2059, many current methods are unable to provide high yields or allow a diverse set of nucleoside triphosphates to be made via a combinatorial or high throughput parallel synthesis.
The three phosphate moieties of nucleoside triphosphates are designated α, β, γ, where the α-phosphate is positioned closest to the sugar moiety. α-Phosphate substitutions in nucleoside triphosphate analogues have proven useful in elucidation of enzymatic functions and mechanisms. For example, exchange of one of the α-phosphate oxygens for sulfur, borane or methyl has allowed investigations of the stereochemical course of certain enzymatic reactions. (See, e.g., Eckstein, et al., Ann. Rev. Biochem., 1985, 54, 367-402; Kaizhang, et al., J. Org. Chem., 1998, 63, 5769-5773; and Victorova, et al., Nucleic Acids Research, 1992, 20, 783-789).
Further, nucleoside triphosphates have important therapeutic and diagnostic applications. These molecules have found important roles, for example, in inhibition of viral replication as is characteristic of AIDS chemotherapy (see, e.g., Meyer, et al., The EMBO Journal, 2000, 19, 3520 and Schneider, et al., Nucleosides, Nucleotides & Nucleic Acids, 2001, 20, 297). Nucleoside triphosphates and derivatives thereof have also been found to have promising anticancer, hypolipidemic, antiinflammatory, and antiosteoporotic activities.
Accordingly, preparation of nucleosides and their derivatives has been studied for many years. For example, Simoncsits, et al., Tetrahedron Letters, 1976, 44, 3995-3998, report the synthesis of 5′-α-amino-triphosphates based on the selective replacement of one of the two amide groups of 5′-phosphorodiamidates. Ludwig, Acta Biochim. Biophys. Acad. Sci. Hung., 1981, 16, 131-133, reports a route to nucleoside 5′-triphosphates. α-P modified nucleoside triphosphate analogs are reported in International Publication Number WO 01/14401. Ludwig, et al., J. Org. Chem., 1989, 54, 631-635, reports a method of making nucleoside 5′-triphosphates and α-thio analogues that requires the protection of the ribose 2′ and 3′ hydroxyl groups and produces byproducts that are difficult to remove. Gaur, et al., Tetrahedron Letters, 1992, 33, 3301-3304, disclose a method of making nucleoside 5′-triphosphates and α-thio analogues using a functionalized solid support that serves as an anchor and protecting group throughout the chemical manipulations. Additionally, He, et al., J. Org. Chem., 1998, 63, 5769 reports the synthesis of 5′-α-boranotriphosphates.
Preparation of other derivatized nucleosides and nucleic acids has been studied. For example, Mori. et al., Nucleic Acids Research, 1978, 5, 2945-2957, report phosphoroselenoate nucleic acid derivatives in which selenium replaces one of the phosphate oxygens through substitution of one of the non-bridging oxygen atoms using potassium selenocyanoate as the selenium donor. Stawinski, et al., J. Org. Chem., 1994, 59, 130-136, disclose a method of synthesizing dinucleoside phosphoroselenoates through use of a selenium transferring reagent to convert nucleoside H-phosphonate and H-phosphonothioate diesters into their corresponding phosphoroselenoates and phosphorothioselenoates. Brownlee, et al., Nucleic Acid Research, 1995, 23, 2641-2647, report a solid phase method of synthesizing 5′-diphosphorylated oligoribonucleotides in preparing capped oligonucleotides. Nyilas, Tetrahedron Letters, 1997, 38, 2517-1518, disclose a one-pot synthesis of γ-amidite modified nucleoside triphosphates by opening cyclic trimetaphosphate with different amines. Krzyzanowska, et al., Tetrahedron, 1998, 54, 5119-5128, adapted the Ludwig, 1989, supra, method to create a one-pot procedure for synthesizing 2′-deoxyribonucleoside 5′-(α-P-borano)triphosphates.
As pharmacological agents, nucleoside triphosphates are often difficult to employ because of the hydrolytic lability of the triphosphate group. As a result, these compounds have short half lives in biological systems and often decompose too quickly to be of therapeutic value. Modified nucleoside triphosphate compounds, such as those that mimic natural analogs and have desired downstream pharmacological effects, are current pharmaceutical goals. In particular, nucleosides having modifications that increase hydrolytic stability, maintain biological activity, and exhibit low toxicity are currently desired. More efficient methods for their synthesis are also needed.